Killer cell immunoglobulin-like receptors (KIRs) belong to immunoglobulin superfamily and are expressed on both natural killer (NK) cells and a subset of T cells. Based on the number of extra-cellular domains, KIR genes are classified as KIR2D and KIR3D. Depending on the length of the cytoplasmic tail and the presence or absence of immunoreceptor tyrosine-based inhibitory motif (ITIM), KIRs can be functionally divided into inhibitory KIR (iKIR) and activating KIR (aKIR). KIR receptors regulate NK cell activities and convey activating or inhibitory signal through interaction with class I human leukocyte antigen (HLA) ligands, which play an important role in transplantation, elimination of tumor cells and resistance to viral infection through innate immune pathways.
The KIR gene cluster on human chromosome 19 consists of 14 functional KIR genes (KIR2DL1, 2DL2, 2DL3, 2DL4, 2DL5, 2DS1, 2DS2, 2DS3, 2DS4, 2DS5, 3DL1, 3DL2, 3DL3, 3DS1) and two pseudogenes (KIR2DP1 and 3DP1) (1). The structure of KIR genes is extremely complicated. Except that KIR3DL3 lacks exon 6, other functional KIR3D genes including 3DL1, 3DL2, and 3DS1 possess 9 exons, 8 introns, a 5′-promoter region and a 3′-untranslated region (3′-UTR). Exons 1 and 2 encode leader peptide. Exons 3, 4 and 5 encode the extra-cellular domains D0, D1 and D2, respectively. Exon 6 encodes the stem. Exon 7 encodes the transmembrane region. Exons 8 and 9 encode the cytoplasmic region. Among the KIR2D genes, KIR2DL1˜3 and 2DS1˜5 have an untranslated pseudoexon 3, which results in the absence of the corresponding extra-cellular domain D0. KIR2DL4 and 2DL5 are characterized by the complete absence of exon 4, and therefore their protein product has no extra-cellular domain D1.
The full genomic sequences for all the KIR alleles released in the IPD-KIR database varies from 9901 bp to 17009 bp in size (2). However, the coding sequences (CDS) of each functional KIR gene has a total length of only 915˜1368 bp (see Table 3), which imply that the non-coding sequence (8773˜15641 bp) accounts for the majority of full genomic sequences of KIR gene (see Table 4). Particularly, the length of introns 5 and 6 accounts for 44.0%˜61.2% of the full genomic sequence of corresponding KIR gene (also see Table 4).
Both exon 1 (either 34 or 40 bp) and exon 2 (36 bp) of each functional KIR gene are short in length and have limited single nucleotide polymorphism sites (SNPs). KIR2DL2, 2DL4 and 2DS4 lack SNPs in both exons 1 and 2, whereas other functional KIR genes possess 1˜3 SNPs, respectively. Thus, routine sequence-based typing at exons 1 and 2 is not required for each KIR gene. In addition, intron 1, which is located between exon 1 and exon 2, is 199˜2280 bp in length. Polymerase chain reaction (PCR) amplificon covering the entire exon 1, exon 2 and the intervening intron 1 will be moderate in length for each KIR gene and can be amplified effectively.
Exon 3, exon 4 or exon 5 of each functional KIR gene is relatively long in length (282˜300 bp) and has much SNPs. Since pseudoexon 3 for KIR2DL1˜3 and 2DS1˜5 doesn't required to be detected, PCR amplification covering the entire exon 4, intron 4 and exon 5 can be achieved in a single amplicon using one pair of KIR gene-specific PCR primers, and then sequencing of exons 4 and 5 needs to be performed separately in both directions for these 8 functional KIR genes. Likewise, both KIR2DL4 and 2DL5 miss exon 4, PCR amplication covering the entire exon 3, intron 3/4 and exon 5 can be achieved in a single amplicon using one pair of KIR gene-specific PCR primers and then sequencing of exons 3 and 5 needs to be performed separately in both directions. As for the other four functional KIR genes (KIR3DS1, 3DL1˜3), which all possess exons 3, 4 and 5, the PCR amplication covering the entire exon 3, intron 3, exon 4, intron 4 and exon 5 can be achieved in a single amplicon using one pair of KIR gene-specific primers and then the sequencing of exons 3, 4 and 5 needs to be performed separately in both directions.
Apart from KIR3DL3 without exon 6, the exon 6 of all the other functional KIR genes is only 51 bp in length. According to the IPD-KIR Database (Release 2.6.0), KIR2DS4, 3DL1, 3DL2, and 3DS1 genes lack SNPs in exon 6, other functional KIR genes possess 1˜2 SNPs. The distribution of SNPs located in exon 6 of each KIR gene is limited. However, the flanking intronic sequences of exon 6 which include introns 5 and 6 have a total length of up to 4937˜9841 bp (see Table 4). To ensure the effective PCR amplication, the entire intronic sequences of intron 5 and/or intron 6 should be avoided in case of generating extreme long PCR amplicon. Thus, the target sequence of exon 6 for each KIR gene should be amplified separately in a single amplicon if necessary.
The length for exons 7, 8 and 9 of each functional KIR gene is 102˜105 bp, 51˜53 bp and 8˜270 bp, respectively. The length for introns 7 and 8 is 460˜462 bp and 98˜118 bp, respectively. Therefore, PCR amplification covering entire exon 7, intron 7, exon 8, intron 8, and exon 9 can be performed in a single amplicon and will not generate ultra-long PCR fragment.
The structural features of full genomic sequence for all functional KIR genes, the characteristic of SNPs distribution in coding sequence and the length of each exon and its flanking intronic sequence that we have mentioned above, are critical to develop a scientific and efficient PCR amplification strategy.
KIR genes exhibit extensive diversity in both haplotype content and allelic diversification. So far 698 KIR alleles including 7 null alleles have been released in the IPD-KIR Database (Release 2.6.0). Among the 14 functional KIR genes, KIR3DL2 exhibits the highest level of allelic diversity with 112 different identified alleles (see Table 5).
Identification of KIR alleles can carry functional significance. McErlean et al. (3) have found that mRNA expression level for the 14 functional KIR genes varies with the hierarchy KIR3DL2>KIR2DS2>KIR3DS1>KIR2DS5>KIR2DL5>KIR2DS3>KIR2DL1>KIR3DL1>KIR2DS1>KIR2DL2>KIR2DL4>KIR2DS4>KIR2DL3. Even within the same KIR gene, the expression level on NK cell surface, the affinity to cognate ligand and the capacity of medicated inhibition or activation can be influenced by different allele. It has been reported by Yawata et al. (4) that the expression levels of KIR3DL1 alleles were in the order of KIR3DL1*01502>*020>*001>*007>*005, whereas the levels of 3DL1-mediated NK cell inhibition were in the order of KIR3DL1*001>*005>*01502>*020>*007. KIR3DL1*005 combines low cell surface expression with a high inhibitory capacity. KIR3DL1*004, a most common KIR3DL1 allele in Caucasians, is poorly expressed at the cell surface (5). KIR2DS4*003, *004, *006, *007, *008, *009, *010, *012, and *013 alleles have a 22 bp deletion at coding sequence (CDS) nucleotide position nt454˜nt475 in exon 5, which causes a reading frame shift, yielding a truncated KIR2DS4 protein with loss of the transmembrane and cytoplasmic domains. These deleted variants of KIR2DS4 protein can't be anchored to the cell surface (6). Thus, it is critical important to identify allelic variation within the 14 functional KIR genes, especially those common null alleles. Since KIR allelic variation alters the level of protein expression and the affinity for cognate ligand as well as the mediated inhibitory/activating capacity, it is an urgent task to develop a low-cost, high-throughput, simultaneous sequence-based typing (SBT) method and apply the established SBT method in KIR-associated disease studies.
To date, the widely-used polymerase chain reaction-sequence specific primer (PCR-SSP) and PCR-sequence specific oligonucleotide probe (PCR-SSOP) commercial kits can only identify the presence or absence of KIR genes on low-resolution level, but can not identify all the KIR alleles at the allele level, especially for those null alleles.
Sequence-based typing is a powerful technique for KIR genotyping at allele level. However, there are no commercial KIR SBT kit and corresponding software for KIR allele assignment in worldwide until now. As KIR genes share extensive sequence homology, it is difficult to design KIR gene-specific primers for PCR amplification of target sequences. While summarizing the characteristic of the KIR SBT methods in the previously published literatures, several problems existed in: {circle around (1)} Only exons encoding extra-cellular domains and/or cytoplasmic region were sequenced for some KIR genes (7, 8, 9). Since the entire coding sequence was not sequenced and the diversity of each exon could not be allowed for analyzing, which led to being prone to generate ambiguous allele combination in SBT test. {circle around (2)} The PCR amplicons could cover the entire coding sequence of each KIR gene, however, the fragments size of PCR amplicon were extremely too long. For example, the KIR3DS1 amplicon covering exon 3 through 3′ untranslated region (3′-UTR) generated a fragment of approximate 12.2 kb in length, and PCR extension at 68V required up to 13 min in each cycle (10), as a result the total PCR amplification time exceeded 10 hours, more high requirement for DNA quality as well as the high-fidelity DNA polymerase were needed in PCR amplification. {circle around (3)} As KIR genes share extensive sequence homology, non-specific amplification or co-amplification occurred in PCR procedure. e.g., while amplifying the target sequence covering exon 1 through exon 5 of 2DL1 in a subject carrying both 2DL1 and 2DS1 genes, 2DS1 would also be co-amplified (11). To obtain 2DL1 specific PCR products, the secondary amplification needed to be carried out using nested PCR primers, which made the PCR procedure cumbersome. {circle around (4)} Due to the different annealing temperatures for PCR primers and varied extension time in each PCR cycling, PCR amplifications could not be carried out simultaneously under the same thermocycling parameters while amplifying the target sequences of 14 functional KIR genes (10, 11, 12, 13), which made PCR procedure more time-consuming and labour-consuming. {circle around (5)} Identification of the KIR alleles with one or more base pair insertion/deletion by traditional cloning and sequencing could not allow for the desired rapidity and simplicity in routine KIR genotyping.
With the elucidation of biological functions for KIR molecules, the clinical significance of the increasingly recognized KIR polymorphism and its role played in transplantation and disease associated studies have drawn extensive interest. Therefore, establishment of the method for high-throughput simultaneous sequence-based typing of 14 functional KIR genes, together with its commercialization and industrialization are currently urgent problems to be solved.